Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Complement C5a-mediated chemotaxis is preserved in Gnaq/Gna11 double knockout and Gna12/Gna13 double knockout macrophages. A, schematic diagram highlighting genes of the Gαq/Gα11 (Gnaq and Gna11) and Gα12/Gα13 (Gna12 and Gna13) families of Gα subunits that potentially may be activated by C5aR. B, summary box plots of cell velocity and chemotactic efficiency (chemotaxis index). *p < 0.05; Kruskal–Wallis test and post-hoc Mann–Whitney U test with Bonferroni correction (n = 75 for each group; 3 independent experiments). C, migration plots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. D, 200 × 300-μm snapshots of WT, Gnaq/Gna11 dKO, and Gna12/Gna13 dKO macrophages in a chemotactic complement C5a gradient. Black arrows, elongated trailing ends. The schematic diagram on the left shows a μ-Slide Chemotaxis chamber with one of the two 40-μl reservoirs (filled with a blue dotted pattern) containing 20 nm complement C5a. E, box plots of maximal tail lengths developed by macrophages migrating in a chemotactic complement C5a gradient over a 6-h period. *, p < 0.05; Kruskal–Wallis test and post hoc Mann–Whitney U test with Bonferroni correction (n = 50 cells/group; sampled from two independent experiments). F, representative example, from two independent experiments, of RhoA activity measured using a colorimetric G-LISA assay, in which active RhoA (RhoA-GTP) was indexed as absorbance at 490 nm (A490). RhoA protein was used as positive control. Bars, mean ± S.D. (error bars) of duplicate measurements.

Article Snippet: Levels of active RhoA (RhoA-GTP) were measured using a colorimetric RhoA G-LISA activation assay kit (catalog no. BK124, Cytoskeleton, Denver, CO), according to the elaborate instruction manual.

Techniques: Chemotaxis Assay, Double Knockout, MANN-WHITNEY, Migration, Activity Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Knockout mouse models reveal the contributions of G protein subunits to complement C5a receptor–mediated chemotaxis

doi: 10.1074/jbc.RA119.011984

Figure Lengend Snippet: Tabular summary and schematic diagram of G protein subunits involved in transducing complement C5a gradients into directed migration. A, tabular summary of results. B, schematic summary. C5aRs couple (i) directly to at least two heterotrimeric G proteins formed by Gα15 and Gαi2 subunits and possibly also Gα12/Gα13 and Gαi3 (not shown) subunits and their respective Gβγ subunits and (ii) indirectly to Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP signaling, which stimulates P2Y2Rs. The Gαi2 subunit is indispensable for chemotaxis and associates with Gβ2-containing, or possibly also Gβ1-containing, Gβγ subunits. Gαi2/Gβ2γx heterotrimeric G proteins, where x is unknown, dissociate into active (GTP-bound) Gαi2 subunits and Gβ2γx dimers following receptor activation by complement C5a. The Gβ2γx (or possibly Gβ1γx) dimers activate PI3Ks, which catalyze the conversion of PIP2 to PIP3. PIP3 is known to recruit pleckstrin homology domain–containing Rac- and Cdc42-GEFs to the membrane. Activation of Gα15-containing heterotrimeric G proteins directly by complement C5a, as well as indirect activation of Gαq/Gα11-containing heterotrimeric G proteins via autocrine ATP and UTP signaling, increases the activity of PLC-β isoforms, which catalyze the hydrolysis of PIP2 to inositol IP3 and diacylglycerol. IP3 induces Ca2+ release from the endoplasmic reticulum, but this Ca2+ signal is largely redundant for lamellipodial membrane protrusions and chemotaxis. However, we speculate that depletion of PIP2 by PLC-β isoforms and PI3Ks contributes to the formation of lamellipodial membrane protrusions by promoting the dissociation of Rac– and Cdc42-GTPase–activating proteins (GAPs). We speculate that activation of Gα12/Gα13 by complement C5a-C5aR signaling, which remains to be confirmed, increases the activity of the monomeric (small) G proteins RhoA and RhoB via RhoGEFs. Activated (GTP-bound) RhoA and RhoB promote actomyosin-dependent retraction of the trailing end of migrating cells, whereas the RhoGAP Myo9b is thought to inhibit RhoA and RhoB at the front of cells. Extracellular ATP and UTP stimulate P2Y2Rs. ATP, but not UTP, additionally activates P2X receptors (not shown), ligand-gated cation channels. ATP and UTP are rapidly degraded by surface ectonucleotidases, such as CD39, to form ligands for other purinergic receptors (not shown).

Article Snippet: Levels of active RhoA (RhoA-GTP) were measured using a colorimetric RhoA G-LISA activation assay kit (catalog no. BK124, Cytoskeleton, Denver, CO), according to the elaborate instruction manual.

Techniques: Migration, Chemotaxis Assay, Activation Assay, Activity Assay

Journal: Redox Biology

Article Title: RhoA GTPase phosphorylated at tyrosine 42 by src kinase binds to β-catenin and contributes transcriptional regulation of vimentin upon Wnt3A

doi: 10.1016/j.redox.2020.101842

Figure Lengend Snippet: P -Tyr216 GSK-3β stimulates β-catenin in early period and then p -Ser9 GSK-3β attenuates β-catenin through Src phosphorylation in late period of Wnt3A stimulation. (A) HEK293T cells were stimulated by Wnt3A in time-dependent manner and indicated proteins were determined. (B) RhoA-GTP levels were determined using RBD-beads pull-down assay. (C) HEK293T cells were transfected with si-GSK3β and reconstituted with GSK-3β WT, S9A, S9D, Y216E and Y216F. Cells were stimulated by Wnt3A (30 ng/ml) for 2 h and indicated proteins were determined. (D) Recombinant GST-GSK-3β WT, S9A, S9D, Y216E and Y216F proteins were incubated with Src protein in the presence of ATP (20 μM) and MgCl 2 (25 mM) for 60 min and p -Ser and p -Tyr416 Src were detected with western blotting. (E) HEK293T cells were stimulated with Wnt3A (30 ng/ml) for 2 h and Src was immunoprecipitated and then indicated proteins were detected. (F) Amino acids sequences of N - (NTD) and C -terminal domains (CTD) of Src, β-catenin and glycogen synthase (GS) were compared. Identical amino acids were indicated in blue and similar ones were noted in cyan blue. Specific Ser candidate residues to be phosphorylated were noted in red. Particularly identical proline residues were noted in green. (G) GST-GSK-3β S9D and Src proteins were incubated with ATP/MgCl 2 , the SDS-PAGE was performed, and Src protein was analyzed to identify phosphorylation site by using MALDI-TOF. Ser51 was identified to be phosphorylation site. (H) Domains of Src including SH4, Unique domain (UD), SH3, SH2, and SH1 was presented. P -Ser51 and p -Ser43 were localized in UD and p -Ser493 was localized in SH1 domain. (I) Cells were transfected with Src S493A, S51A, S43A mutants and WT (wild type), then Src was immunoprecipitated and then p -Ser and p -Tyr416 Src, p-S9 GSK-3β, p -Tyr216 GSK-3β, p -Tyr42 RhoA, ROCK1, and ROCK2 were immunoblotted. (J) Cells were transfected with Src S493D, S51D, S43D mutants, and WT (wild type), then Src was immunoprecipitated and then p -Ser and p -Tyr416 Src, p-S9 GSK-3β, p -Tyr216 GSK-3β, p -Tyr42 RhoA, ROCK1 and ROCK2 were immunoblotted. (K) Cells were transfected with Src A3 (S493A/S51A/S43A), D3 (S493D/S51D/S43D), A2 (AA: S493A/S51A), D2 (DD: S493D/S51D), AD (S493A/S51D) and DA (S493A/S51A), and WT (wild type) and Sc was immunoprecipitated and then p -Ser and p -Tyr416 Src, p-S9 GSK-3β, p -Tyr216 GSK-3β, p -Tyr42 RhoA, ROCK1 and ROCK2 were immunoblotted. (L) si-GSK-3β, GSK-3β WT and GSK-3β S9A were transfected and treated with Wnt3A for 2 h. Src was immunoprecipitated and p -Ser was immunoblotted. (M) Tentative model for p -Ser9 GSK-3β to phosphorylate Ser493, Ser51, and Ser43 residues of Src was presented. p -Ser493 and p -Ser51 Src are inactive forms, but p -Ser43 Src is an active form. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Equivalent amounts of protein from the supernatants were then incubated with GST-Rhotekin-RBD for GTP-RhoA (Pierce).

Techniques: Pull Down Assay, Transfection, Recombinant, Incubation, Western Blot, Immunoprecipitation, SDS Page

Journal: International Journal of Clinical and Experimental Pathology

Article Title: The protective effect of the Rho-kinase inhibitor hydroxyfasudil on propofol-induced hippocampal neuron apoptosis in neonatal rats

doi:

Figure Lengend Snippet: Hydroxyfasudil improves the tendency towards cognitive impairments induced by propofol. Evaluation of rat learning and memory in the Morris water maze. A. Mean escape latency to the platform in the Morris water maze. B. Time spent in platform quadrant among the different groups. C and D. Pull-down experiments for GTP-bound RhoA in primary culture hippocampal neurons with indicated treatment. Data were shown as the mean ± SD, **P < 0.01 vs. the control group; #P < 0.05, ##P < 0.01 vs. the “P” group.

Article Snippet: The RhoA activation assay was performed to detect the endogenous active form of RhoA (RhoA-GTP) using a RhoA activation assay kit (Abcam; ab211164) following the manufacturer’s manuals.

Techniques:

Journal: Journal of Cellular Physiology

Article Title: Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation

doi: 10.1002/jcp.27910

Figure Lengend Snippet: Protein expression of Gal‐3, integrin β1, RhoA, GTP‐RhoA in AS and some paired normal segments. (a) ELISA was used to detect the level of Gal‐3 in plasma. (b) Protein expression of Gal‐3, integrin β1 and GTP‐RhoA was assessed by WB and the relative protein expression was further indicated using histograms. ** p < 0.01 versus control

Article Snippet: Subsequently, the membranes were incubated with the following primary antibodies at 4°C overnight: integrin β1 (1:1000, cat.24693, abcam), RhoA (1:1000, cat.6352, Affinity), phosphorylation of RhoA (GTP‐RhoA) (1:500, cat.211164, abcam), JNK (1:1000, cat. AF6319, Affinity), p‐JNK (1:1000, cat. AF3320, Affinity), ICAM‐1 (1:1000, cat. DF7413, Affinity) and VCAM‐1 (1:1000, cat.DF6082, Affinity), NF‐κB P65(1:1000, AF5006, Affinity), PhosphoNFκB P65(1:1000, AF2006, Affinity), IKKα (1:1000, AF6012, Affinity), IKKβ (1:1000, bs‐4880R, Bioss, China), Phospho‐IKKβ(1:1000, bs‐3232R, Bioss, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular Physiology

Article Title: Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation

doi: 10.1002/jcp.27910

Figure Lengend Snippet: Gal‐3 increases the expression of integrin β1, GTP‐RhoA and p‐JNK in ox‐LDL induced HUVECs. After exposure to Gal‐3, ox‐LDL or their combination, the expression of integrin β1 was detected by IF, (a) WB was used to measure the protein levels of integrin β1, RhoA, JNK, GTP‐RhoA, and p‐JNK (b). The relative protein expression was further indicated using histograms (c). ** p < 0.01, ## p < 0.01 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Subsequently, the membranes were incubated with the following primary antibodies at 4°C overnight: integrin β1 (1:1000, cat.24693, abcam), RhoA (1:1000, cat.6352, Affinity), phosphorylation of RhoA (GTP‐RhoA) (1:500, cat.211164, abcam), JNK (1:1000, cat. AF6319, Affinity), p‐JNK (1:1000, cat. AF3320, Affinity), ICAM‐1 (1:1000, cat. DF7413, Affinity) and VCAM‐1 (1:1000, cat.DF6082, Affinity), NF‐κB P65(1:1000, AF5006, Affinity), PhosphoNFκB P65(1:1000, AF2006, Affinity), IKKα (1:1000, AF6012, Affinity), IKKβ (1:1000, bs‐4880R, Bioss, China), Phospho‐IKKβ(1:1000, bs‐3232R, Bioss, China).

Techniques: Expressing

Journal: Journal of Cellular Physiology

Article Title: Galectin‐3 exacerbates ox‐LDL‐mediated endothelial injury by inducing inflammation via integrin β1‐RhoA‐JNK signaling activation

doi: 10.1002/jcp.27910

Figure Lengend Snippet: Gal‐3 aggravates ox‐LDL induced HUVECs injury by activating integrin β1‐RhoA‐JNK pathway. HUVECs, treated by ox‐LDL alone or in combination with Gal‐3, were performed to knockdown integrin β1 by siRNA, and the levels of integrin β1 and relative factors (JNK, pJNK, RhoA and GTP‐RhoA) were examined by WB and demonstrated by histogram (A and B). IF was performed to assess the expression of integrin β1 after knockdown. (c) Cell viability was measured by MTT assay (d) and apoptosis was determined by flow cytometry (E and F) using siRNA or JNK inhibitor SP600125. The changes of JNK and p‐JNK expression were evaluated after the treatment of RhoA inhibitor Y‐27632 alone or in combination with Gal‐3. (G and H) ** p < 0.01, ## p < 0.01 [Color figure can be viewed at wileyonlinelibrary.com]

Article Snippet: Subsequently, the membranes were incubated with the following primary antibodies at 4°C overnight: integrin β1 (1:1000, cat.24693, abcam), RhoA (1:1000, cat.6352, Affinity), phosphorylation of RhoA (GTP‐RhoA) (1:500, cat.211164, abcam), JNK (1:1000, cat. AF6319, Affinity), p‐JNK (1:1000, cat. AF3320, Affinity), ICAM‐1 (1:1000, cat. DF7413, Affinity) and VCAM‐1 (1:1000, cat.DF6082, Affinity), NF‐κB P65(1:1000, AF5006, Affinity), PhosphoNFκB P65(1:1000, AF2006, Affinity), IKKα (1:1000, AF6012, Affinity), IKKβ (1:1000, bs‐4880R, Bioss, China), Phospho‐IKKβ(1:1000, bs‐3232R, Bioss, China).

Techniques: Expressing, MTT Assay, Flow Cytometry